To begin, inject a preemptive analgesic into an anesthetized mouse. Using clippers, shave the head of the mouse. Install the animal on a stereotaxic table and correctly position ear bars.
Attach the mouse's mouth to the mouse piece and carefully position the tongue outside to prevent suffocation. After checking the depth of anesthesia, position elevation support beneath the animal to ensure the body and head remain leveled horizontally. And use a warm pad with paper to maintain the animal's temperature.
Apply ointment to both eyes to prevent dryness. Then, using iodine and alcohol, disinfect the mouse's head. Use a scalpel to make an incision along the head's midline with the cut stretching from behind the eyes to post the lambda suture.
Expose the skull and swab it with a sterile cotton tip saturated in 0.9 sodium chloride solution. Under a stereo microscope, identify the rostral rhinal vein or RRV and use a sterile pencil to mark it. Using a sterile needle as a reference, confirm the orientation of the brain.
Ensure no change in the DV and ML coordinates. Next, position a sterile glass pipette containing the virus solution to the RRV reference for zero anteroposterior reference. Advance the pipette along the sagittal suture until reaching the chosen anteroposterior coordinate.
Mark this spot with a sterile pencil and lift the needle. Carefully drill a small circle around the marked position to avoid breaking the superior sagittal sinus, and remove the piece of the skull with small forceps. Then, using the superior sagittal sinus median as the mediolateral reference, move the pipette to the position and lower it until it touches the dura mater as the dorsoventral reference.
Create a slight break in the dura mater and lower the pipette to the preferred dorsoventral position. Inject the specified quantity of adeno-associated virus or AAV vectors into the brain and remove the pipette. Free the mouse from the ear bars, then close the skin using surgical clips.
transition the animal into a separate warmed cage to recover. Keep an eye on the mouse's recovery. Once it regains consciousness, return it to its original enclosure when fully recovered.
